Intrathecal [64Cu]Cu-albumin PET reveals age-related decline of lymphatic drainage of cerebrospinal fluid

Age-related cognitive decline is associated with dysfunctional lymphatic drainage of cerebrospinal fluid (CSF) through meningeal lymphatic vessels. In this study, intrathecal [64Cu]Cu-albumin positron emission tomography (PET) was applied in mice to evaluate lymphatic drainage of CSF and its variation with age. [64Cu]Cu-albumin PET was performed at multiple time points after intrathecal injection of [64Cu]Cu-albumin at an infusion rate of 700 nl/min in adult and aged mice (15–25 months old). CSF clearance and paravertebral lymph nodes were quantified after injection and during the stationary phase. Stationary phase of the next day followed the initial perturbed state by injection of 6 ul (1/7 of total CSF volume) and CSF clearance half-time from the subarachnoid space was 93.4 ± 19.7 and 123.3 ± 15.6 min in adult and aged mice (p = 0.01), respectively. While the % injected dose of CSF space were higher, the activity of the paravertebral lymph nodes were lower in the aged mice on the next day. [64Cu]Cu-albumin PET enabled us to quantify CSF-lymphatic drainage across all levels of brain spinal cords and to visualize and quantify lymph node activity due to CSF drainage. [64Cu]Cu-albumin PET revealed the age-related decrease of the lymphatic drainage of CSF due to this decreased drainage from the subarachnoid space, especially during the stationary phase, in aged mice.

enough to cover the range of adulthood but separate from the other at least 6 months difference. This could have reduced variability-related type II error (false negative results).

Sample size
For ethical reasons, we tried to reduce the number of mice as small as possible, but for acquisition of the sufficient number to discover the true difference between groups, we needed to increase the number of mice as large as affordable (in terms of the survival of the mice after intrathecal injection and repeated anesthesia/sedation and imaging in warm blanket within the gantry of PET scanner).
Sample size was determined by the prior experiment which established the imaging protocol and injection/anesthesia/mice wellbeing and survival techniques (18).

Inclusion and exclusion criteria
Considering that the repeated imaging procedures after the initial anesthesia/operation/intrathecal injection preclude any possibility of exclusion, the only reason of exclusion was technical failure of experiment despite meticulous handing of entire experimental/imaging procedures. 15-25 months old mice of aged group were the survivors till that age, and thus the survivors in our institutional animal facility taken care of by ourselves and thus survivors. The mice which failed to thrive would have been excluded.

Randomization
Adult mice were randomly selected among the mice we obtained from vendor, and those who survived the aging were also included to the aged group.

Blinding
Blinding between adult and aged mice group could not be blinded to the investigators. Blinding of reading mice PET and of quantification based on drawing of regions of interest (ROIs) on CSF or organs were not done simply because it was not relevant. Retrospectively, the same ROI was transferred from the first image labeled 'immediate' to the images of later time (2h, 4h, … 24h).
However, the ROIs should have been adjusted due the position change of mice at each successive imaging epoch.

Outcome measures
Two measures are relevant to say as for outcome measures. First, alternation scores for Y-maze were measured as established standard. Especially acclimation the day before and the measurement ensured the reproducibility and thus the variability of alternation scores per individual mice represent the biological variability of individual mice. Second, individual variability of CSF ROI activity were quite large to obliterate the difference between groups at earlier times (immediate, 2h, 4h) but came to be mitigated at later time (especially 24h).

Statistical measures
Statistical methods using non-parametric comparison of adult and aged groups of mice of seven/six could yield sufficiently low type I error (false positive). As we have measured individual mice sixtimes repeatedly, entire number of measurement was 78, meaning 78 volume-type whole body PET images available for several ROI quantification. As this study was exploratory one that the hypothesis was that there would be difference between adult and aged mice and as we found the difference in CSF clearance and lymph node uptakes along time after intrathecal injection, we now can make posterior hypothesis that there might be difference between aged normal controls and young disease (for example, AD model such as 5xFAD) model mice with of without novel treatment. In this further experiment, we might use lymph node to CSF ratio at 24h PET as a parameter to reveal the effect of novel disease-modifying treatment.

Experimental animals
We used a single strain normal mice whose strain was C57BL/6 and only male mice. This was because our contingent experiment was about the reduced lymphatic drainage of CSF in male 5xFAD mice. 5xFAD mice are derivative of C57BL/6 strain. Female mice and their aging-related effect on CSF-lymphatic drainage is open to further investigation.

Experimental procedures.
Technical establishment was reported in our previous publication and in the main text, especially for the handling of radiopharmaceutical and radioligand for labeling and stability in mice in vivo after intrathecal injection.

Results
Details of results are referred to the main text. To avoid redundancy and to maintain succinctness of description we would not comment any further here in this supplementary note.